1. Field of the Invention
The present invention is directed to mucosal immunity. Specifically, the invention is directed to a method of inducing mucosal immunity in a subject. More specifically, the invention is directed to a method of inducing mucosal immunity in a subject by administering DNA complexed to a lipospermine to the mucosa of the subject.
2. Background Art
Mucosal surfaces represent the major route of entry for most systemic pathogens with subsequent mucosal immunity usually providing long-term protection against reinfection (25). Examples include,the life-long immunity produced by the Sabin oral polio vaccine versus the relatively short-term protection provided by,the Salk parenteral vaccine (48) and the single dose oral cholera vaccine with its improved safety profile versus the older multi-dose parenteral cholera vaccine (27). The best long-term mucosal and systemic protection against infection is provided by live, attenuated pathogens which simulate infection of the naive host but which are incapable of inducing disease (28). Despite the current capacity to produce attenuating mutations in cloned microorganisms, the concern over potential reversion to virulence or host virulence determinants has effectively inhibited development of live attenuated pathogens as. inducers of mucosal immunity for human use (29).
For example, at the most recent meeting of the NIH sponsored HIV vaccine meeting in November 1994, the proponents of attenuated live virus vaccines received a blow by Ruth Ruprecht (29) who reported that an attenuated SIV (i.e., Nef deletion)was responsible for the development of AIDS in newborn Rhesus macaques who had received the vaccine. It is unlikely that an attenuated HIV will ever receive FDA approval as an HIV vaccine.
The World Health Organization (WHO) estimates that by the year 2000 at least 40 million people, will be infected with the Human Immunodeficiency Virus (HIV). Due to the relentless and progressive pathogenesis of the virus the majority of those infected will-die within 10 years. It is estimated further that the death toll will be at 10 million as we enter the 21st century. Despite an initial massive effort by industry to develop a vaccine, few commercial developers remain. NIH""s National HIV Vaccine recently received a critical blow when the AIDS Research Advisory Program Committee (ARAC) voted to not proceed in Phase III clinical testing of the two leading candidate subunit vaccines.
Another difficulty with the current efforts to develop an HIV vaccine is the paucity of research in the generation of mucosal immune responses to HIV. Epidemiological data clearly indicate that 70-80% of all AIDS cases are the result of heterosexual transmission of HIV (30-38). Heterosexual transmission is the fastest. growing route of transmission in the United States with women being at significantly greater risk of infection by HIV than males (39,40). Since 90% of HIV is transmitted sexually worldwide, it is unlikely that systemic-immunity will block initial infection at the mucosal sites of entry. Infection of Langerhans cells, mucosal macrophages, T cells, and even epithelial cells from cell associated HIV or free HIV in semen of the genital tract is a powerful argument that the induction of mucosal responses are at least as important as systemic responses in the development of a vaccine against HIV infection (35-37,41). Although systemic immunization rarely induces mucosal immunity, mucosal immunization frequently provides systemic responses as well (36,41,42,43,44,45,46). It is essential that more effort be devoted to this key element in establishing a primary defense against, HIV transmission. With the clear danger of using live attenuated virus, the prospects for inducing mucosal immunity are difficulty.
Recent developments in vaccine research include the demonstration that transfection of mouse muscle with a bacterial plasmid carrying the DNA sequence encoding an influenza virus nucleoprotein resulted in the development of humoral and cellular responses which protected mice from lethal viral challenge (1). Two methods of transfection in vivo have been reported previously to achieve genetic immunization. The more common approach follows the observation that mouse muscle is a unique, target for transfection with naked DNA (3) and that muscle of a variety of species is particularly susceptible to naked DNA transfection (4-11). Protection against lethal challenge in mice by influenza A virus, and induction of cytotoxic lymphocytes and neutralizing antibodies to influenza A virus (1,13-15) and HIV (16,17), following genetic IM immunization has been reported by a number of investigators. Despite the impressive induction of protective immune responses, this method has the disadvantage that relatively massive quantities of DNA are required. Although unreported as a toxic side effect to date, this requirement for large quantities of DNA may limit this method due to the potential for antibody response to DNA itself and the generation of a self-sustaining lupus-like syndrome. The less common approach to genetic immunization using bolistic transformation overcomes the problem of DNA quantity but requires instrumentation not widely available. Typically, nanogram quantities of DNA complexed to gold or tungsten particles are physically propelled through the plasma membrane by microprojectile bombardment. Both methods elicit cellular (21,22) and humoral responses (22-24). However, neither of the above methods of genetic immunization induce mucosal immunity.
Despite the importance of mucosal immunity for an effective immunization strategy, the only FDA approved vaccine that induces mucosal immunity is the Sabin, live-attenuated oral polio vaccine. More recently, another development in the generation of mucosal immunity was the demonstration that the systemic administration of activated vitamin D3 (1,25-dihydroxycalciferol [1,25(OH)2D3]) with conventional protein antigens converts a systemic response to a mucosal response (2). Thus, the art is actively seeking ways to induce a mucosal immune response.
The present invention meets a very important need in vaccine production by providing a method to induce in vivo mucosal immune responses to antigens of pathogens by the facilitated transfection of mucosa with a bacterial plasmid carrying the DNA sequence for the antigen.
The invention provides a method of inducing a mucosal immune response in a subject, comprising administering to the mucosa of the subject an amount of antigen-encoding DNA effective to induce a mucosal immune response complexed to a transfection-facilitating lipospermine or a lipospermidine. In the method of inducing a mucosal immune response, the antigen-encoding DNA can encode an antigen that is expressed on the surface of infected cells during the course of infection. The present method should apply to all mucosally acquired pathogens in which expression of antigen on the surface of a mucosal cell mimics natural infection. DNA encoding the envelope glycoproteins of viral pathogens is the rational choice for use in the present method.
Lipospermines and lipospermidines are bifunctional molecules consisting of one or more hydrophobic chains covalently linked to a cationic grouping in which there is coordination of three or more amide hydrogens with a phosphate oxygen of the DNA chain forming an ionic charge complex. One preferred example of a lipospermine is DOGS (dioctadecylamidoglycylspermine). Diotadecylamidoglycylspermidine is another likely candidate, because it has the same structure as DOGS, but lacks one of the two arms having two non-essential cationic charges.
The invention also provides a composition, comprising an amount of DNA encoding an envelope antigen or envelope-associated antigen of a pathogen complexed to a lipospermine. More specifically, the invention provides a composition, comprising an amount of DNA encoding an envelope antigen of HIV complexed to a lipospermine